KR20060059557A - Method for preparation of dermal papilla tissue inducing hair folicle formation - Google Patents

Abstract

The inventive method for the preparation of a dermal papilla tissue in accordance with the present invention makes it possible to form a quantity of the dermal papilla tissues having hair follicle inductive ability, and accordingly, it can be effectively used for the treatment of alopecia through cell transplantation.

Description

METHODS FOR PREPARATION OF DERMAL PAPILLA TISSUE INDUCING HAIR FOLICLE FORMATION}             

1 is a diagram schematically showing the structure of hair follicles,

2 is a schematic diagram showing a method for separating the complete hair follicles of the growth phase from the skin of a mammal (rat),

3 is a view showing a process of primary culture by separating the dermal papilla and the lower tuberculosis cells from the separated hair follicles,

4 is a photograph taken when the cells of the primary papilla and lower tuberculosis were passaged successively.

5 is a photograph showing a process of inducing self-aggregation of cultured dermal papilla cells to form cell aggregation, where A is hair follicle dermal constituent cells (capillary papilla and lower tubercle weed) cultured in a culture vessel at least 90%, B is aggregation Cells cultured for at least 3 weeks after changing to medium for C, C is a shape in which the migrated cells are gradually aggregated and start to aggregate, D is a cell before aggregation is almost completed and suspended from the culture vessel, and E is a culture vessel. Multiple cell aggregates formed simultaneously in several places in, and F represent cross sections of hematoxylin / eosin histostained aggregates, respectively.

6 is a schematic diagram of an experiment for observing the interaction of hair follicles and hair follicles in Comparative Example 1 and Experimental Example 2,

FIG. 7 is a photograph showing the formation of new hair follicle structures in vitro through the interaction between two tissues when mixed and cultured between collagen gels at a slight distance from actual hair papillae and hairless hair follicles.

FIG. 8 shows a new culture in vitro similar to FIG. 7 after 6 days as a result of mixing and culturing hair follicle dermal constituent cells with tissue (arrow head) made by inducing cell self-aggregation and hair follicles without arrows (arrows). It is a picture taken to form a mogu (arrow head).

The present invention relates to a method for producing dermal papilla tissue capable of inducing hair follicles, and more specifically, after dermal papilla and lower dermal sheath cells isolated from hair follicles are grown in a general cell culture medium. The present invention relates to a method for preparing dermal papilla tissue that plays an important role in the growth and maintenance of hair follicles by culturing in a medium containing high concentrations of amino acids and vitamins and growth factors.

There are various types of hair loss, including male type, female type, and circular baldness. In the past, artificial hair transplantation was used as a proper countermeasure. However, it was prohibited to be used for severe side effects. Currently, the drug treatment method and the hair transplantation of the hair where the hair loss does not occur to the hair loss site Transplantation methods are used. In the case of treatment with drugs, hair loss progresses as soon as the drug is stopped by delaying the progression of hair loss or by helping to maintain the current hair condition. In the case of hair transplantation, the transplanted hair is transplanted to the forehead area by using the hair of the back of the head, which is not bald, and the transplanted hair is a permanent hair with a growth cycle unlike when hair is settled as a complete hair follicle in the graft. . However, it is true that the number of hair transplanted is limited, and in fact, the hair of the head is not infinite, so when about 2,000 hairs are transplanted at a time, it is usually impossible to operate more than 3 times. In addition, current hair transplantation is expensive and involves the collection and transplantation of hair follicles for transplantation and pain in patients for a considerable period after transplantation. Therefore, it is important to develop an appropriate transplant plan according to the overall hair loss progression and hair loss condition.

As such, there are many limitations in the treatment of baldness, and in order to overcome this, studies are being conducted to transplant cells constituting hair follicles in vitro. On the other hand, culturing hair follicles in vitro is also used as a method for studying hair follicle properties.

As a study on the characteristics of conventional hair follicles, Arrays et al. (Arase S. et al., Skin Pharmacol. 7 (1-2): 12-5, 1994) have been described for the removal of hair follicles and hair follicles of isolated human hair follicles. In vitro and incubated together, the hair follicle cells migrated toward the dermal papilla to form new hair follicles.The formation and maintenance of the hair follicles was found between the epidermal outer root root sheath (ORS) cells and the dermal papilla cells. It has been shown that hair follicles can be reconstructed if they are made by complex and strong interactions and only if they are sufficient. In addition, Cohen J., J Embryol Exp Morphol. 9: 117-27, 1961 and Oliver RF., J Embryol Exp Morphol. 15 (3): 331-47, 1966 and Oliver RF., J Embryol Exp Morphol. 18 (1): 43-51, 1967) demonstrated that the dermal papilla plays a decisive role in the growth of hair follicles through microscopic separation and animal transplantation experiments using rat vibrissa.

On the other hand, Reynolds AJ et al., Development. 122 (10): 3085-94, 1996) isolated and cultured dermal papilla cells in vitro, which is a unique feature of dermal papilla cells after about 3-4 generations. Inamatsu M. et al., J Invest Dermatol. 111 (5): 767-75, 1998) reported that the hair follicle induction ability was gradually lost . Rat hair follicle dermal constituent cells were cultured using epithelial lung medium.

Yahoda (Jahoda CA., Development. 115 (4): 1103-9, 1992) and Reynolds AJ. Et al., Development. 115 (2): 587-93, 1992) isolated the mouse sensory dermal papilla. After culturing, cells of three generations or less were transplanted to the ear wounds and the back of rats, and reported to form hairy hairs on the graft site, Gharzi et al. (Gharzi A. et al., Exp Dermatol. 12 (2): 126 -36, 2003) reported that hair follicles are induced even when the outer hair root and lower weaves of rat sensory hair are cultured in collagen gel similar to the actual skin and then implanted with the actual papillae between the two cell layers. It was. Kevin J. et al., J Invest Dermatol. 121: 1267-1275, 2003 also demonstrated that not only the dermal papilla cells but also the underlying tubercle cells surrounding the dermal papilla are capable of inducing hair follicles. Recently, lower tuberculosis cells have also been widely used in the study of hair follicles.

Thus, the present inventors have isolated the hair follicles, dermal papilla and lower dermal sheath cells known to have hair follicle induction ability to grow up to 5 to 6 generations using generally known culture medium, and then In vitro reorganization of dermal papilla tissues, which play an important role in the growth and maintenance of hair follicles, using a medium containing growth factors in a medium containing amino acids and vitamins, thereby self-aggregating cells without a specific support and substrate to produce large amounts of dermal papilla tissue. It has been found that the present invention can be formed to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for in vitro production of dermal papilla tissue to enable self-aggregation of cells without specific support and substrate to form a large amount of papillary tissue.

In order to achieve the above object, the present invention

1) separating the dermal papilla and lower tuberculosis cells from the hair follicles,

2) culturing the isolated cells up to 5 to 6 generations in a cell culture medium each containing amino acids in an amount of 600 to 1,900 mg / L and vitamins in an amount of 12 to 36 mg / L, respectively,

3) The cultured cells were added with a growth factor necessary for the growth and maintenance of hair follicles in a serum-free medium containing amino acids in an amount of 2,000 to 3,000 mg / L and vitamins in an amount of 40 to 60 mg / L, respectively. Incubating the cells to self-aggregate, and

4) provides a method for producing dermal papilla tissue, comprising the step of recovering the self-aggregated and suspended cells in a centrifuge and again added to the culture medium.

The present invention will be described in more detail below.

In the method of the present invention, in culturing hair papilla and lower tuberculosis cells of hair follicles, DMEM (Dubecco's Modified Eagle Medium) medium to which 10% fetal bovine serum (FBS) is added, or lung medium cultured with epithelial cells Unlike conventional methods that use, dermal papilla and lower tuberculosis cells first cultured in a conventional cell culture medium containing amino acids in an amount of 600 to 1,900 mg / L and vitamins in an amount of 12 to 36 mg / L, respectively. Cultured in a medium in which a growth factor necessary for the growth and maintenance of hair follicles was added to a high serum-free medium containing amino acids in an amount of 2,000 to 3,000 mg / l and vitamins in an amount of 40 to 60 mg / l, respectively. It is done.

In the present invention, the lower tuberculosis cells and the dermal papilla cells are separated from the hair follicles of the complete growth phase and primary cultured in a known method of conventional cell culture medium. The cell culture medium is composed of DMEM (Dulbecco's Modified Eagle Medium), DMEM / F-12, F-12, Mc Coy's 5A, RPMI 1640, William Medium E (Williams' medium E), IMDM (Iscove's Modified Dulbecco's Modification), and the like. Can be selected from the group. Primary and continuous cultures allow dermal constituent cells (hair papilla and lower tuberculosis cells) to be cultured up to 5 to 6 generations that are considered to have lost hair follicle induction ability. When primary cultured cells were cultured using high concentration culture medium and growth factor containing 2 to 5 times of amino acid and vitamin compared to the above-mentioned general medium without special support or substrate, about 80% of monolayer cultured cells Aggregates can be formed to reproduce the characteristics of dermal constituent cells. For example, when 1 × 10 ⁶ cells are inoculated into a 25 cm 2 culture vessel, hundreds of self aggregates are formed.

Since the high concentration medium used in the present invention contains 2 to 5 times higher concentration of amino acids and vitamins than the above-mentioned conventional cell culture medium, nutrient deficiency and oxygen generated in the culture of dermal papilla cells and lower tuberculosis cells. Overcomes deficiency and improves the differentiation properties of cells to help form the dermal papilla tissue.

The high concentration DHGM medium of the present invention is an amino acid of L-arginine, L-asparagine, L-aspartic acid, L-cystine 2HCl, L-isoleucine, L-leucine, and L-lysine HCl are each 30 To 200 mg / l, the aromatic amino acids L-phenylalanine, L-tryptophan, and L-tyrosine of the essential amino acids each containing 30 to 210 mg / l, and the rest of the essential amino acids each containing 50 to 600 mg / l. It is preferable.

In addition, as vitamins, biotin, D-Ca pantothenate, and riboflavin, which are water-soluble vitamin B groups, were 0.02 to 1 mg / l, respectively, and choline chloride, folic acid, niacinamide, pyridoxine HCl, and thiamine HCl were respectively 3 to 16 mg / l. L, i-inositol preferably contains 10 to 15 mg / l, and vitamin B ₁₂ contains 0.02 to 0.03 mg / l. In addition, the medium of the present invention more preferably contains 0.03 to 0.07 mg / L of glutathione, 400 to 600 mg / L of glutamine, and 1,500 to 3,000 mg / L of D-glucose.

In addition, sodium bicarbonate (NaHCO ₃ ) 3,000 to 3,500 mg / l with pH buffering effect, and Hepis (HEPES, N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid]) 2,000 to 2,500 mg / l, Ca and Mg, minerals important for cell-to-cell adhesion, at concentrations of 1.0 to 2.0 mM and 0.5 to 1.0 mM, respectively, Cu, Fe, Mn, and Zn, other minerals that are important for cell metabolism It is more preferable to contain at a concentration of 0.25 to 0.7 nM each, and to include sodium chloride at a concentration of 4,000 to 5,000 mg / l in order to adjust the osmotic pressure to 280 to 310 mOsm / kg.

In addition, the medium of the present invention is added hydrocortisone (HC), insulin (I), transferrin (T), and sodium selenite (S) to further extend the culture period of hair follicles For example, hydrocortisone is 10 to 100 μg / L, insulin is 5 to 20 mg / L, transferrin is 5 to 20 mg / L, and sodium selenite is 0.005 to 0.02 mg / L It is preferable to use at the concentration of.

In particular, in the present invention, CaCl ₂ 165 mg / L, CuSO ₄ · 5H ₂ O 0.0001 mg / L, Fe (NO ₃ ) .9H ₂ O 0.0001 mg / L, KCl 330 mg / L, KNO ₃ 0.076 mg / L, MgSO ₄ 98 mg / l, MnCl ₂ · 4H ₂ O 0.0001 mg / l, NaCl 4,800 mg / l, NaHCO ₃ 3,360 mg / l, Na ₂ HPO ₄ 111 mg / l, ZnSO ₄ · 7H ₂ O 0.0002 mg / l, D-glucose 2,000 mg / l, sodium pyruvate 110 mg / l, HEPES 2,383 mg / l, phenol red 15 mg / l, L-alanine 50 mg / l, L-arginine 100 mg / l, L-asparagine 50 Mg / l, L-aspartic acid 60 mg / l, L-cystine.2HCl 182.4 mg / l, L-glutamic acid 150 mg / l, L-glutamine 584 mg / l, L-glycine 60 mg / l, L-histidine 62.1 mg / l, L-isoleucine 210 mg / l, L-leucine 210 mg / l, L-lysine HCl 292 mg / l, L-methionine 60 mg / l, L-phenylalanine 132 mg / l, L-proline 80 mg / l, L-serine 84 mg / l, L-threonine 190 mg / l, L-tryptophan 32 mg / l, L-tyrosine 208 mg / l, L-valine 188 mg / l, biotin 0.026 Mg / l, D-Ca Pantothe Yitrate 0.026 mg / l, choline chloride 8 mg / l, folic acid 16 mg / l, i-inositol 14.40 mg / l, niacinamide 8 mg / l, pyridoxine HCl 8 mg / l, riboflavin 0.8 mg / l, thiamine HCl 3 Preferred is a medium consisting of mg / l, and vitamin B ₁₂ 0.026 mg / l.

The preferred high-concentration medium used in the present invention has a two-fold increase in amino acids and vitamins that supply energy for cell metabolism and maintain cell activity, and because it is metabolized to lactic acid, it has a lesser role as an energy source in living bodies compared to the general medium. The content of D-glucose is reduced to 2,000 mg / l. The concentration of L-glutamine affecting the amount of ATP synthesis in the amino acid is fixed at 4 mM. In addition, the trace elements include zinc 0.7 nM, iron 0.25 nM, copper 0.4 nM, and manganese 0.5 nM, and sodium bicarbonate and HEPES having a pH buffering effect at concentrations of 40 mM and 10 mM, respectively. Final concentrations of calcium and magnesium, which are essential minerals for intercellular adhesion, are 1.5 mM and 0.8 mM, respectively, and contain 4,500 mg / l of sodium chloride to adjust the osmotic pressure to 280 to 310 mOsm / kg.

In addition, 10 mg / l of transferrin as an iron source was added, 0.01 mg / l of sodium selenite as an inorganic salt, 10 μg / l of hydrocortisone as a hormone and 10 mg / l of insulin, 0.2% by weight of albumin, and HGF as a growth factor ( Hepatocyte growth factor) 20 ng / ml.

When using serum-containing media in hair follicle organ cultures, hair follicles have a good initial growth rate but tend to prematurely deteriorate and are susceptible to infection, making them unsuitable for long-term culture of hair follicle organs (Randall VA. Et al., J Investig). Dermatol Symp Proc. 8 (1): 39-45, 2003) Although it is difficult to analyze the data, the medium of the present invention does not include serum and thus is useful as a medium for culturing and storing hair follicle organs since there is no fear of infection.

In addition, the important pH in cell culture is kept constant by the buffering effect between bicarbonate and the end metabolites. That is, the medium of the present invention may have a higher concentration of amino acids than William medium E, which may cause a pH increase due to the metabolite ammonia, but the pH is well maintained between 7.2 and 7.5 due to the addition of bicarbonate buffer.

Copper sulfate contained in the culture medium for hair follicles of the present invention inhibits apoptosis due to radical ions through superoxide dismutase (SOD), a copper-dependent enzyme and antioxidant present in hair follicles. It is known to inhibit both 5α-reductase-1 and -2 in hair follicles through dependent enzymes, thereby promoting natural growth factor synthesis and promoting the synthesis of extracellular matrix. Zinc is also known as an essential mineral for zinc finger transcription factors.

The dermal papilla self-aggregates prepared according to the present invention are approximately 100-200 μm in size and have direct cell interactions because they are similar to the actual papillae and use natural cell contact, and the contact is via hematoxylin / eosin tissue staining. Looking at the cross section, the cells are precisely aggregated. In particular, aggregates do not require any support for external stimulation or cell attachment and proliferation, and can be produced in large quantities and have sufficient hair follicle induction ability to be useful for the treatment of baldness through cell transplantation and the study of hair follicle characteristics in vitro. Can be used.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the invention only.

Preparation Example 1 Preparation of High Concentration Medium

According to the composition shown in Table 1, 1 L of a medium having an increased amino acid and vitamin content compared to the conventional medium was prepared, and in this medium, insulin 10 mg / L, transferrin 10 mg / L, sodium selenite 0.01 mg / L , 10 μg / L hydrocortisone, and 0.2 wt% (2 g / L) albumin were added.

Example 1 Isolation of Hair Follicles

Hair follicles were obtained by surgical operation of the vibrissa hair of Spague-Dawley rats. The isolated sensory hairs were stored in tubes filled with William's medium E (Gibco BRL, N.Y., U.S.A.), a common hair follicle culture medium. Skin containing hair follicles was washed in a PBS solution containing antibiotics penicillin G (10 units / ml), streptomycin (10 μg / ml) and amphotericin B (25 μg / ml), and then using a scalpel. Hair follicles were obtained by removing the surrounding adipose tissue and dermis tissue. Among the hair follicles separated by light microscopy, the hair growth stage was selected as much as possible, and the growth cycle was determined based on the structure of hair follicles. The separated growth phase hair follicles were micromanipulated to remove the remaining adipose tissue and dermal tissue using a needle of a 1 cc syringe to separate only the complete growth phase hair follicles (FIG. 2).

Example 2: Isolation and Primary Culture of Breast Teat and Lower Tuberculosis

First, 400 μl of fetal calf serum was added to a 35 mm cell culture dish so that the dermal papilla tissue was well attached, and the bottom of the container was evenly wetted. And looking at the separated hair follicle tissue through an optical microscope, using a syringe needle to remove the dermal papilla in the tissue, and separate the lower tuber sheath surrounding the outer papilla, put each in a culture vessel moistened with fetal bovine serum in a 37 ℃ incubator I put it. When each tissue was attached to the bottom of the culture vessel, the culture medium DMEM (Dulbecco's Modified Eagle Medium; Gibco BRL) was added. At this time, the fetal calf serum was initially adjusted to be 20% in consideration of the fetal calf serum initially added to increase the adhesion of the tissue. The medium was added carefully and no medium was replaced until cells flowed out of the tissue. Once the cells started flowing out, the medium was changed to DMEM with 10% fetal calf serum (FIG. 3).

Example 3: Preparation of Breast Papillary Tissue

The primary papilla and lower tuberculosis cells cultured in Example 2 were subsequently cultured using DMEM containing 10% fetal calf serum (FIG. 4). When cultured up to 5 to 6 generations or more, in which papillary and lower tuberculosis cells almost lost hair follicle induction ability, cells were monolayered to about 90% of the culture vessel (FIG. 5, A). Thereafter, the culture medium was changed to a serum-free medium prepared in the same manner as in Preparation Example 1 above, and the growth factor HGF (rhHGF, R & D system) was added at a concentration of 20 ng / ml. (Figure 5, B). The medium was replaced about once every 5 days. Cell aggregation was observed from about 4 weeks after the culturing (Fig. 5, C), and the aggregated cell mass was naturally suspended from the culture vessel into the medium (Fig. 5, D and E). From the time point at which the cell aggregates were suspended, the waste medium was collected when the medium was replaced, and the cell aggregates were recovered by centrifugation at 500 rpm for 3 minutes. If not used immediately in the experiment was suspended in a fresh medium and put in a culture vessel to maintain the culture state.

As can be seen from the results of D and E of Figure 5, the dermal papilla self-aggregates according to the production method of the present invention is about 100-200 ㎛ in size similar to the actual dermal papilla, because direct cell interaction is used strong.

Experimental Example 1: Confirmation of cohesion of dermal papilla tissue

In order to confirm the cohesion of the dermal papilla tissue prepared by the method of Examples 1 to 3, the contact cross section was examined through known hematoxylin / eosin tissue staining.

As a result, as can be seen in F of Figure 5, the cells were also precisely aggregated, it also showed robustness not easily broken by external stimulation.

Comparative Example 1: Hair follicle formation by actual dermal papilla

As shown in FIG. 6, the collagen gel was filled with about 700 μl in a 24-well plate and gelled by placing it in a 37 ° C. incubator for 1 hour. The hair papilla separated from the method of Example 2 and the hair follicle from which the hair follicles were removed were placed at a distance of about 300 μm, incubated for about 5-10 minutes, and adhered to the gel surface. Then, the collagen solution was again covered with about 700 μl. It was then gelled and culture medium K-SFM (Gibco BRL, NY, USA) was added. After about a week, the outer hair root sheath cells were induced to surround the dermal papilla, and after about 15 days, the induced cells were more numerous, and the shape of the hair follicle structure was more pronounced (FIG. 7).

Experimental Example 2: Hair follicle formation by the prepared papillary tissue

In order to see if the dermal papilla tissue prepared in Example 3 can actually induce hair follicles, the experiment was conducted in the same manner as in Comparative Example 1 except that the dermal papilla tissue of Example 3 was used.

As a result, it was observed that the hair papilla tissue prepared in the present invention and the cells of the hair follicles from which the hair follicles were removed interact with each other to begin to flow out of the hair follicles to form new hair follicle-like tissues after about a week (FIG. 8). ).

Therefore, it can be seen that the dermal papilla tissue prepared according to the present invention has the ability to induce hair follicles as well as the actual dermal papilla.

By using the method of preparing the dermal papilla tissue of the present invention, the dermal papilla tissue having hair follicle inducing ability can be prepared in sufficient amount through the proliferation of cells in vitro, and thus the method of the present invention can be used to study the characteristics of the hair follicles and explant the cells in vitro It can be usefully used to treat baldness.

Claims (8)

1) separating the dermal papilla and lower tuberculosis cells from the hair follicles, 2) culturing the isolated cells in a cell culture medium each containing amino acids in an amount of 600 to 1,900 mg / L and vitamins in an amount of 12 to 36 mg / L, and 3) The cultured cells were added with a growth factor necessary for the growth and maintenance of hair follicles in a serum-free medium containing amino acids in an amount of 2,000 to 3,000 mg / L and vitamins in an amount of 40 to 60 mg / L, respectively. Self-aggregating the cells by culturing in Method for producing a dermal papilla tissue comprising a. The method of claim 1, In step 3), the method of producing a dermal papilla tissue further comprises the step of recovering the self-aggregated and suspended cells and added to the culture medium again. The method of claim 1, In step 2), the medium for cell culture is Dulbecco's Modified Eagle Medium (DMEM) medium, DMEM / F-12, F-12, Mc Coy's 5A, RPMI 1640 medium, Williams E medium, and IMDM ( Iscove's Modified Dulbecco's Modification) A method for producing dermal papilla tissue, characterized in that it is selected from the group consisting of medium. The method of claim 1, In step 2), the method of producing a dermal papilla tissue, characterized in that the cells are cultured for 5 to 6 generations. The method of claim 1, In step 3), wherein the growth factor is hepatocyte growth factor (hepatocyte growth factor) method for producing dermal papilla tissue. The method of claim 1, The serum-free medium of step 3) contains L-arginine, L-asparagine, L-aspartic acid, L-cystine 2HCl, L-isoleucine, L-leucine, and L-lysine HCl, respectively, as amino acids. At mg / l, L-phenylalanine, L-tryptophan, and L-tyrosine at 30-210 mg / l, respectively, and L-alanine, L-glutamic acid, L-glycine, L-histidine, L-methionine, L- 50-600 mg / l of proline, L-serine, L-threonine, and L-valine, respectively; Biotin, D-Ca pantothenate, and riboflavin as vitamins, respectively, at 0.01 to 2 mg / l, choline chloride, folic acid, niacinamide, pyridoxine HCl, and thiamine HCl at 3 to 16 mg / l, respectively, i-inositol At 10 to 15 mg / l and vitamin B ₁₂ at 0.02 to 0.03 mg / l; Glutamine 400-600 mg / l, D-glucose 1,500-3,000 mg / l, sodium bicarbonate (NaHCO ₃ ) 3,000-3,500 mg / l, and hepis (HEPES, N- [2-hydroxyethyl] piperazine-N '-[ 2-ethanesulfonic acid]) containing 2,000 to 2,500 mg / l, Ca 1.0 to 2.0 mM, Mg 0.5 to 1.0 mM, Cu, Fe, Mn, Zn each 0.25 to 0.7 nM, and sodium chloride 4,000 to 5,000 mg / l. Method for producing a dermal papilla tissue, characterized in that. The method of claim 1, The serum-free medium of step 3) further comprises insulin, transferrin, sodium selenite, hydrocortisone, and albumin. The method of claim 1, The dermal papilla tissue is an aggregate composed of the dermal papilla and the lower mycorrhizal cells.

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